Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
The function of the immune response is to disarm invading pathogens or toxins. The immune response can in some circumstances be very destructive to an organism and survival depends upon the ability of the immune system to distinguish self from non-self. Autoimmune diseases, for example, develop when the immune system over responds to self. Some immune responses are against non-self molecules that are relatively harmless. Asthma and hayfever, for example, involve immune responses to non-self where the immune response is more debilitating than the causative agent. Generally, the innate immune system screens out responses to non-pathogenic organisms and helps to prevent adaptive immune responses to such harmless agents.
Adaptive immune responses are carried out by lymphocytes such as B lymphocytes that carry out antibody responses or T lymphocytes which carry out cell mediated responses. B lymphocytes produce immunoglobulins which help to deactivate pathogens and toxins. T cells react directly with non-self molecules (antigens) that are presented on the surface of host cells in association with major histocompatibility (MHC) molecules that provide a repertoire of “self” molecules. In both cases, a cellular response is generated that is specific to particular epitopes of the non-self molecule and provides a network of immune responses and immune effector molecules.
Accordingly, one method for diagnosing or monitoring an infection or evaluating the ability of a subject to mount an immune response to non-self is to determine whether the subject has mounted an immune response to antigen stimulation. As the T cell response comprises the production of effector T cells that are capable of responding to an antigen or can be stimulated to respond to the antigen by producing immune effector molecules, one can measure the production of these molecules in vitro in response to specific antigens as a measure of a cell mediated immune response. However, as non-self antigenic molecules are presented to T-cells by antigen presenting cells there is a complex interaction of molecules and cells that must take place successfully in vitro in order to produce sufficient immune effector molecule for detection.
Most in vitro methods for detecting cell mediated immune responses involve the purification of peripheral blood mononuclear cells from whole blood using various separation techniques. Such assays include chromium release assays, cytotoxicity assays, MHC class I tetramer assays, assays for IFN-γ or other cytokines, of which ELISPOT provides a good example. The ELISPOT method immobilizes antigen presenting cells and has been used to detect the number of T-cells producing certain cytokines in response to antigenic stimulation.
If whole blood is used, it is generally diluted in a culture medium in order to dilute red blood cells, which are considered to reduce the sensitivity of the assays. An in-tube cell-mediated immune response assay which uses undiluted whole blood is described in International Publication No. WO 2004/042396 in the name of Cellestis Limited incorporated herein in its entirety by reference. International Publication No. WO 2004/042396 discloses the use of blood collection tubes for the incubation of sample with antigen and a simple sugar and shows enhanced sensitivity using the tube system compared to assays where blood is transferred to and incubated in 24-well microtitre plates.
For whole blood assays in humans and livestock animals, at least about three milliliters of blood is taken from the subject in order to provide sufficient material to perform cell-mediated immune response assays. This amount is generally taken by venous blood sampling, via needle into a collection vessel, often under vacuum.
Various methods of detecting immune effector molecules, such as enzyme-linked immunosorbent assay (ELISA), radio-immuno assay (RIA), or cytometric methods can use small volumes, however, there is a need in the art for improved systems for conducting the antigenic stimulation phases of in vitro cell-mediated immune response assays. In particular, methods that allow whole blood testing in small volumes of blood such as those obtained by peripheral capillary sampling are needed. The ability to screen small samples of blood would greatly facilitate sampling of children or other subjects where blood may be limited or difficult to obtain, and allows blood sampling without venous blood sampling by using capillary blood such as that obtained by prick testing of the thumb, heel, ear-lobe or other convenient site, and for testing multiple or a range of antigen including mitogen and hapten stimulants in a single blood draw of low volume.